MALDI Biotyper System Research Articles

Evaluation of MALDI-TOF mass spectrometry coupled with ClinProTools as a rapid tool for toxin-producing Clostridioides difficile

IntroductionThe performance of MALDI-TOF MS combined with analysis platform for identification of toxin-producing Clostridiodes difficile is yet to be known. MethodsBetween August 2018 and September 2020, 61 isolates from stool specimens of patients with C. difficile-associated diarrhea were analyzed using the MALDI Biotyper system. A C. difficile toxin-producer detection model was developed using ClinProTools. The model was validated using 28 known strains that differed from the isolates used to develop the model. ResultsThe sensitivity and specificity of the Genetic Algorithm (GA) model using isolates grown on Brucella with hemin and vitamin K (BHK) agar plates were 91.7% and 44.4%, respectively. When isolates grown on cycloserine-cefoxitin mannitol agar were analyzed by the model, sensitivity and specificity were 6.3% and 100%, respectively. The GA model using BHK medium showed the highest discriminatory performance in detection of toxin-producing C. difficile. However, a discrepancy in detection of toxin-producing C. difficile was observed in the results generated when the model was being developed and when the model was validated which suggests that incubation conditions may have affected the results. ConclusionMALDI-TOF analysis using ClinProTools has a potential to be a cost-effective tool for rapid diagnosis and contribute to antimicrobial stewardship by differentiating toxin-producing C. difficile from non-producers.

Improved identification of Streptococcus bovis-Streptococcus equinus-complex species and subspecies by MALDI-TOF MS using a novel library

ObjectivesTo develop an in-house matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) library for improved identification of species and subspecies of the Streptococcus bovis/Streptococcus equinus-complex (SBSEC). MethodsA total of 236 SBSEC isolates from blood stream infections and culture collections, determined by whole genome sequencing to subspecies level, were grown in brain heart infusion broth. Mass spectra were collected using the Bruker MALDI Biotyper system after ethanol-formic acid extraction. Main spectral profiles from 117 isolates were used to create the “SBSEC-CMRS library.” The remaining 119 spectra were used for evaluation of Bruker MALDI Biotyper (MBT) Compass Library Revision K (2022) and the SBSEC-CMRS library. ResultsThe Bruker library correctly identified species and subspecies in 72 of 119 (61 %) isolates, while the SBSEC-CMRS library identified 116 of 119 (97 %), using a cutoff score of ≥2.0. ConclusionsThe SBSEC-CMRS library showed sufficient diagnostic accuracy, and can be implemented in clinical practice for SBSEC species and subspecies identification.

Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n=1; T.interdigitale, n=2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

Molecular Confirmation of an Indole-Negative Klebsiella oxytoca Isolated from a Patient with Cystitis.

Klebsiella oxytoca is an opportunistic pathogen that causes nosocomial infections. Here, we describe an unusual clinical strain of indole-negative K. oxytoca, GU175, isolated from the urine of a patient with cystitis. The GU175 strain was identified as K. pneumoniae with a probability of 99.40%, negative for indole production, and resistant to third-generation cephalosporins by using the MicroScan Walkaway 40 SI system with the Negative combo EN1 J panel. Biochemical characterization of this strain using lysine-indole motility medium was negative for indole production. However, identification tests using the MALDI Biotyper system and 16S rRNA gene sequence analysis revealed that GU175 is K. oxytoca. DNA sequence analysis of the tryptophanase operon comparing the GU175 strain with the revertant GU176 strain, which tested positive for indole, revealed a point mutation in the Shine-Dalgarno sequence upstream of tnaC in the GU175 strain. This is the first report of indole-negative K. oxytoca, which was attributed to a mutation in the DNA sequence of the tryptophanase operon isolated from a patient with a urinary tract infection. As indole-negative K. oxytoca can be misidentified as K. pneumoniae by biochemical characterization, clinical microbiologists should be aware of such misidentifications.

Comparison of MALDI-TOF mass spectrometry and rpoB gene sequencing for the identification of clinical isolates of Aeromonas spp.

Aeromonas spp., widely present in rivers and soil, cause mild gastroenteritis, severe septicemia, and soft tissue infections in humans. Treatment of these infections require accurate identification of pathogenic Aeromonas spp. However, identification at the species level using conventional methods is highly challenging. In this study, we aimed to compare the accuracy of two different approaches developed for bacterial identification: (i) housekeeping gene sequencing (rpoB) in conjunction with phylogenetic analysis and (ii) matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS) (MALDI Biotyper and VITEK MS), for differentiating Aeromonas spp. We analyzed 58 Aeromonas isolates recovered from patients at different medical institutions in Japan using both identification methods. The rpoB sequencing method was the most accurate, identifying all Aeromonas isolates at the species level. Meanwhile, the MALDI Biotyper system correctly identified 53 (91.4%) isolates at the genus level and an additional 30 (51.7%) at the species level. The VITEK MS system correctly identified 58 (100%) isolates at the genus level and an additional 34 (58.6%) at the species level. Thus, MALDI Biotyper and VITEK MS accurately identified isolates at the genus level, but differences were found in the accuracy of identification of species. However, the low cost and ease of analysis make MALDI-TOF MS-based methods strong candidates for use in clinical laboratories that require easy-to-use identification methods.

The challenges in the identification of Escherichia coli from environmental samples and their genetic characterization

Escherichia coli bacteria are an essential indicator in evaluations of environmental pollution, which is why they must be correctly identified. This study aimed to determine the applicability of various methods for identifying E. coli strains in environmental samples. Bacterial strains preliminary selected on mFc and Chromocult media as E. coli were identified using MALDI Biotyper techniques, based on the presence of genes characteristic of E. coli (uidA, uspA, yaiO), as well as by 16S rRNA gene sequencing. The virulence and antibiotic resistance genes pattern of bacterial strains were also analyzed to investigate the prevalence of factors that may indicate adaptation to unsupportive environmental conditions and could have any significance in further identification of E. coli. Of the strains that had been initially identified as E. coli with culture-based methods, 36–81% were classified as E. coli with the use of selected techniques. The value of Cohen’s kappa revealed the highest degree of agreement between the results of 16S rRNA gene sequencing, the results obtained in the MALDI Biotyper system, and the results of the analysis based on the presence of the yaiO gene. The results of this study could help in the selection of more accurate and reliable methods which can be used in a preliminary screening and more precise identification of E. coli isolated from environmental samples.

Accuracy of ASTA MicroIDSys, a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system, for the identification of Korean reference and clinical bacterial and yeast strains

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control.

Brucellae are intracellular sneaky bacteria and they can elude the host’s defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

Sensitivity of vaginal and mammary bacterial microflora of sows from patients with postpartum dysgalactic syndrome to antibacterial drugs

Purpose: Investigation of the sensitivity of conditionally pathogenic and pathogenic causative agents of postpartum disgalacting syndrome isolated from the pathological discharge of the vagina and the secretion of the mammary glands of patients with sorts to the most common antibacterial drugs.Materials and methods. The fence of the biological material was carried out within 2-3 days after supporting in sows of different ages and parity with the clinical manifestation of postpartum disgalacting syndrome from June to August 2021. In sows, the purse was taken in a mosper, milk, discharge from the vagina for the purpose of bacteriological research.Samples of vaginal wasches were sent in a special transportation environment of Ames. Samples of colostrum and milk (3-5 ml) were gained in sterile test tubes in compliance with the rules of antiseptics (the mammary glands were laid with warm water and 70% ethyl alcohol were treated). For the allocation and study of pure cultures of microorganisms from the above biomaterials produced crops on various nutrient media.The material brought to the laboratory was studied as follows: from the transport medium was carried out primary sowing on triptica-soybean agar, tryptichase-soybean broth, triptichase-soy agar with the addition of 5% of the defibrous blood of the ram; Samples were incubated in aerobic conditions at 37; The growth was taken into account after 24 hours. Then, pure cultures were isolated for the study of the cultural and morphological properties of the microorganisms obtained.The primary identification of the strains of microorganisms was carried out using the Microflex® LRF Bruker Maldi Biotyper system. The accuracy of the results obtained was confirmed by classical microbiological methods based on morphological, cultural and biochemical signs of microorganisms.The resistance of the isolated and identified pure crops to antibiotics was determined by diffusion in agar. In the study, the "advanced set of disks to estimate the antibiotic sensitivity of enterobacteria was used.Results. Inflammatory processes in the reproductive path of sows with postpartum diskalactic syndrome are caused by predominant gram-negative microorganisms, in lactic glands - associations of gram-positive and gram-negative microflora. The main conditionally pathogenic and pathogenic pathogens of endometritis and / or mastitis associated with postpartum diskalactic syndrome are the microorganisms of Escherichia, Enterobacter, Klebsiella, Actinobacillus, Rothia, Weisella, Pseudomonas, Proteus, Enterococcus, Streptococcus and Staphylococcus. It has been established that the overwhelming majority of microorganisms are sensitive to cephalosporine antibiotics: cefepim, zefisim, cefotaxim, ceftazidim, ceftriaxone, ceftinibuthen, cefuroxime.Conclusion. With respect to a large number of the most frequently used antibacterial drugs on this pig-breeding enterprise, high resistance is observed in microorganisms. Treatment of postpartum diskalactic syndrome sowers using antibacterial drugs is recommended to be carried out taking into account sensitivity to them allocated conditional and pathogenic microorganisms.

Reprocessing effectiveness of gastrointestinal endoscopes in western Algeria hospitals: Infectious risk and biofilm formation

The significant increase in endoscopic procedures can lead to a potential source of cross-infection accompanied by biofilm formation on the devices when the disinfection procedure is unsuitable. This study, the first in Algeria, aims to describe the microbiological quality of gastroscopes used in gastroin-testinal units of western Algeria hospitals and to implement some corrective measures. A total of 132samples from endoscopes were examined over a period of 2 years. Contamination level of samples was evaluated by CFU count. Identification of the isolates was made by MALDI Biotyper System. Biofilm formation was detected by Congo Red Agar and tissue Culture Plate method (TCP). The endoscope-drying effectiveness was analyzed by bacterial count and Scanning Electron Microscopy (SEM). Microbial growth was de-tected in 68.18%of endoscopes; 150strains were isolated: Gram-negative-bacilli (56%), Gram-positive-cocci (26.66%), Gram-positive-bacilli(17.33%); dominant species were Enterobacter cloacae, coagulase-negative Staphylo-cocci, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus. Forty one out of 132 samples collected after disinfection disclosed Enterobacteriaceae (41.93%), Pseudomonaceae (16.12%), Staphylococci (30.64%) and Bacillus (11.29%). Using TCP, 96 isolates were good biofilm-producers. The drying reduced significantly the rate of CFU growth for all channels with absence of indicators bacteria. Drying had a better inhibition on biofilm formation, SEM observation revealed a very low abundance of biofilms. Our study is the first in Algeria to describe microbiological findings of post-reprocessing-gastroscopes. Biofilms inside endoscope result in de-contamination failure. Rigorous cleaning/disinfection and drying with com-pressed air are essential elements that reduce the contamination level and biofilm formation inside endoscopes.

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

CryptoType - Public Datasets for MALDI-TOF-MS Based Differentiation of Cryptococcus neoformans/gattii Complexes.

Yeasts of the Cryptococcus neoformans/gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.

Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n=2) and Cryptosporidium hominis (n=1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.

First report of bacterial leaf spot on Chenopodium quinoa caused by Pseudomonas syringae in Colombia

Quinoa (Chenopodium quinoa) has become a food of great importance in the last decade, and it is considered by the Food and Agriculture Organization (FAO) as the grain of the twenty-first century. This plant is considered as a pseudo-cereal with high protein content and digestibility, as well as balanced content of essential amino acids, therefore having multiple beneficial properties for patients with obesity, dyslipidemia and diabetes. Even though this plant can grow under different abiotic and extreme biotic conditions, its production can be affected by diverse pathogens. Here, we show the pathogenicity of Pseudomonas syringae, a Gram-negative bacillus, by isolating pathogenic strains of P. syringae from leaves of C. quinoa cv. piartial with induced necrotic spots at 2–5 days post-inoculation. The bacteria was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a Bruker Daltonik MALDI Biotyper system. Our results constitute the first report in Colombia of the pathogenic P. siryngae in quinoa.

Identification of Infectious Diseases Patterns in the Combined Use of Bacteriological Diagnostics and MALDI Biotyper

In multidisciplinary hospitals, there are conditions conducive to the emergence of healthcare-associated infections: high concentration of people with reduced immunity in a limited area, the presence of a significant number of sources of contagion (patients and carriers), a change in the biocenosis of the mucous membranes and skin of patients and medical personnel under the influence of widespread use of antibiotics and cytostatics. The aim of the research was in the intercomparison of the standardized bacteriological algorithms and the MALDI Biotyper system in the microbiological diagnosis of pathogens as illustrated by the healthcare-associated diseases. Materials and methods . Seventy-eight patients of a multidisciplinary hospital of a regional level (Irkutsk) in 2018–2019 were examined. The age of patients ranged from 1 to 15 years. The material for the study was blood, sputum, swabs from the tracheobronchial tree, throat, nose, wound, abdominal fluid, cerebrospinal fluid, and swabs from environmental objects. Identification of the isolated cultures (78 bacterial strains) was carried out using generally accepted bacteriological methods, as well as using the MALDI Biotyper system. Results and discussions. In the structure of healthcare-associated infections, Pseudomonas aeruginosa occupied a leading position. Not all isolates of microorganisms were identified by standardized bacteriological methods. The identification of strains with characteristic manifestations of physiological and biochemical characteristics was more reliable. Identification difficulties arose in the presence of atypical properties of microorganisms when the use of MALDI Biotyper would be crucial. Conclusion . It is necessary to apply an integrated approach to conduct reliable diagnostics of pathogens. It includes standardized bacteriological methods and methods for identifying microorganisms using mass spectrometry in the subsequent stages.

Rapid animal species identification of feta and mozzarella cheese using MALDI-TOF mass-spectrometry

The correct labelling of foodstuffs with regard to the value-determining ingredients is decisive for the consumer. Therefore, one of the main topics of official food control for meat, seafood and milk products is the inspection of the declared animal species. The use of unlabeled cow's milk products to substitute highly-priced sheep's or buffalo's milk cheese has concerned consumer protection laboratories for many years. Recently, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been demonstrated to identify the animal species of milk used for production of cheese and some other dairy products. The identification was conducted by comparison of mass spectra resulting from samples with those in a corresponding reference database. In this study, a MALDI-TOF MS database for animal species identification of dairy products was created using Bruker's MALDI Biotyper-system in combination with a simple extraction method without a tryptic digestion step. The database contains more than 150 reference spectra of milk and cheese from different dairy species. Using feta/white cheese as well as mozzarella, two of the most consumed cheeses from goat, sheep, cow's or buffalo's milk, we demonstrated MALDI-TOF MS to be an easy, fast and specific tool to identify the animal species of the milk these cheeses were made from. This method has been validated and established in the routine analysis of official food control. Exchange of reference spectra generated and used in this study is facilitated by the MALDI-user platform (https://maldi-up.ua-bw.bwl.de).

Antimicrobial susceptibility of multi-drug and extensively-drug-resistant Escherichia coli to ceftolozane-tazobactam and ceftazidime-avibactam: An in vitro study*

Aim: <i>Escherichia coli</i> is one of the Gram-negative bacteria, known to cause many nosocomial infections. Multi-drug (MDR) and extensively-drug resistant (XDR). <i>E. coli</i> are of particular note, due to significant limitations in antibiotic therapy. Ceftolozane-tazobactam and ceftazidime-avibactam are novel therapeutic options against Gram-negative bacteria; hence the aim of this study was to evaluate and compare the <i> in vitro </i> activity of ceftolozane-tazobactam and ceftazidime-avibactam against MDR and XDR clinical <i>E. coli</i> isolates. Material/Methods: The study included 100 non-replicate <i>E. coli</i> isolates derived from clinical samples of patients hospitalized in teaching hospitals. Bacteria were identified by applying mass spectrometry in the MALDI Biotyper system (Bruker). ESBL (bla<sub>CTX-M-1group</sub>, bla<sub>CTX-M-9group</sub>) and carbapenemase (bla<sub>KPC</sub>, bla<sub>VIM</sub>, bla<sub>NDM</sub>, bla<sub>OXA-48</sub>, bla<sub>OXA-181</sub>) genes were detected using the eazyplex® SuperBug CRE test, based on a loop-mediated isothermal amplification (LAMP). The in vitro susceptibility to ceftolozane-tazobactam and ceftazidime-avibactam was tested using validated MIC Test strips (Liofilchem). Results: All 84 extended-spectrum β-lactamase-producing (ESBL) <i>E. coli</i> isolates were susceptible to ceftazidime-avibactam and 83 to ceftolozane-tazobactam. Among 17 <i>E. coli</i> isolates with resistance to at least one of the carbapenems, three (17.6%) were susceptible to ceftolozane-tazobactam and ceftazidime-avibactam. All 14 blaVIM gene-positive <i>E. coli</i> isolates were resistant to both ceftolozane-tazobactam and ceftazidime-avibactam. Both antibiotics were active against bla<sub>CTX-M-9group</sub> and bla<sub>OXA-48</sub> gene-positive <i>E. coli</i> isolates, but they were not active against bla<sub>CTX-M-1group</sub> and bla<sub>VIM</sub> gene-positive isolates. Conclusions: Ceftolozane-tazobactam and ceftazidime-avibactam are alternative, non-carbapenem therapeutic options for ESBL-positive <i>E. coli</i> strains, and they are promising in the treatment of carbapenem-resistant <i>E. coli</i> strains, but not for those carrying the metallo-β-lactamase enzymes. Both drug combinations have comparable activity against ESBL, however, lower MIC values were found for ceftazidime-avibactam.

Comparing identification of clinically relevant Prevotella species by VITEK MS and MALDI biotyper

In this multicenter study, we aimed to evaluate the performance of MALDI Biotyper and VITEK MS, for identification of Prevotella species. Three hundred and fourteen clinical isolates, collected in eight European countries between January 2014 and April 2016, were identified at the collecting sites by MALDI Biotyper (versions 3.0 and 3.1) and then reidentified by VITEK MS (version 3.0) in the central laboratory. 16S rRNA gene sequencing was used as a standard method. According to sequence analysis, the 314 Prevotella strains belonged to 19 species. MALDI Biotyper correctly identified 281 (89.5%) isolates to the species level and 33 (10.5%) only at the genus level. VITEK MS correctly identified 253 (80.6%) isolates at the species level and 276 (87.9%) isolates at the genus level. Thirty-three isolates belonging to P. bergensis, P. conceptionensis, P. corporis, P. histicola, and P. nanciensis, unavailable in the VITEK MS 3.0 database, were resulted in genus level or no identification. Six Prevotella strains, belonged to P. veroralis, P. timonensis, and P. conceptionensis not represented in the MALDI Biotyper system database, were misidentified at the genus level. In conclusion, both VITEK MS and MALDI Biotyper provided reliable and rapid identification. However, the permanent extension of the databases is needed.

Isolation and characterization of nitrogen removal bacteria from sewage and fecal sludge of dairy cow farms in Cu Chi, Ho Chi Minh City, Vietnam

The bacteria that involved in the removal of nitrogen in wastewater were initially studied. Isolation media were SW-MM (artificial Seawater-Minimal Medium) and 17208 PIA (Pseudomonas Isolation Agar). The pure isolates were streaked on the SW-MM agar plates that added ammonia, nitrate, or nitrite with ascending concentrations to select the isolates had good nitrogen removal potential. The result was that 49 isolates had been collected from 11 samples of sewage and fecal sludge of dairy cow farms, in which including 6 isolates had been isolated by using specialized medium for Pseudomonas. All 49 isolates (100%) were able to grow on the SW-MM added 100 mM ammonia/nitrate or more. The ability to withstand ammonia and nitrate of the best isolates reaches 800 mM. Meanwhile, the ability to withstand nitrite had only belonged to 39 isolates (79.6%) with nitrite concentrations of 5 - 15 mM. Nine isolates were investigated the in vitro ability to reduce ammonia in the wastewater. Quantitative results of ammonia by phenol-hypochlorite method showed that the ability to reduce NH4+ of 9 isolates ranged from 27.9 – 48.9 mg/L on 10 days after inoculation. The six best isolates that had the ammonia- removing efficiency reached 88.5% - 97.5% were identified by using the MALDI Biotyper System (Germany). The identification result showed that XM3, NA6 was Bacillus megaterium, M7C1 was Bacillus pumilus, NA8 was Rhodococcus ruber, MD6 was Pseudomonas fulva, and XM5 was Pseudomonas mendocina. It is recommended to test the ability of these isolates to reduce nitrate and nitrite in the wastewater.

Pseudomonas grimontii, causal agent of turnip bacterial rot disease in Japan

From April to May 2012, a rot disease on turnips grown in greenhouses in southern Hokkaido, Japan caused severe leaf blight, brown discoloration of crown surface, and browning and rot of the interior of main roots. From diseased tissues, bacteria that formed pale beige- to cream-colored colonies were isolated and demonstrated to be pathogenic on turnip after inoculation and reisolation of the isolates. They were gram-negative, aerobic rods with one to three polar flagella, and identified as Pseudomonas grimontii based on biochemical and physiological characterizations, multilocus sequence analysis using concatenated sequences of 16S rRNA, rpoD, gyrB, and rpoB genes, and MALDI-TOF MS analysis using the Bruker MALDI Biotyper system. To the best of our knowledge, P. grimontii has not been isolated previously in Japan, so this study might be the first report of the species in Japan. Although the significance of P. grimontii as a plant pathogenic bacterium has not yet been fully evaluated, we proved in this study that the bacterium is the causal agent of the disease and that its virulence is strong. These findings indicate that it is necessary to record P. grimontii as a novel pathogen posing a threat to plants and requiring vigilance.